ABSTRACT Activating mutations in FGFR3 cause achondroplasia and thanatophoric dysplasia, the most common human skeletal dysplasias. In these disorders, spinal canal and foramen magnum stenosis can cause serious neurologic complications. Here, we provide evidence that FGFR3 and MAPK signaling in chondrocytes promote synchondrosis closure and fusion of ossification centers. We observed premature synchondrosis closure in the spine and cranial base in human cases of homozygous achondroplasia and thanatophoric dysplasia as well as in mouse models of achondroplasia. In both species, premature synchondrosis closure was associated with increased bone formation. Chondrocyte-specific activation of Fgfr3 in mice induced premature synchondrosis closure and enhanced osteoblast differentiation around synchondroses. FGF signaling in chondrocytes increases Bmp ligand mRNA expression and decreases Bmp antagonist mRNA expression in a MAPK-dependent manner, suggesting a role for Bmp signaling in the increased bone formation. The enhanced bone formation would accelerate the fusion of ossification centers and limit the endochondral bone growth. Spinal canal and foramen magnum stenosis in heterozygous achondroplasia patients, therefore, may occur through premature synchondrosis closure. If this is the case, then any growth-promoting treatment for these complications of achondroplasia must precede the timing of the synchondrosis closure.

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Bioarchaeologists rely on accurate estimations of age-at-death. Clearly, some pathological conditions are associated with gross morphological changes in the skeleton that could impact the effectiveness of age-at-death estimation (i.e. methods based on the pelvis, fourth rib, dental attrition, and cranial stenosis). The magnitude of this problem has not been widely studied due to a paucity of pathological skeletons of known age. We assessed age-at death for three individuals affected by bone dysplasias (achondroplasia, residual rickets, osteogenesis imperfecta) using cementum annulations and several osseous age indicators. We predicted osseous indicators that are based on gross morphological changes would yield age estimates discrepant from the cementochronology. Results demonstrated considerable differences in age estimates between morphological and histological techniques suggesting a need for additional research on the effects of pathology on the accuracy of morphological methods. Conversely, we addressed the proposition that cementum annulations will be inappropriate for age estimation in cases of chronic and severe rhino-maxillary infection and periodontitis. We assessed age-at-death for one individual with leprosy and found no indication the disease process affected cementum formation or preservation. The results of this research indicate the potential value of cementochronology in cases where skeletal pathological conditions constrain the usefulness of traditional age estimation approaches.

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Fibroblast growth factors (FGFs) and their receptors (FGFRs) play significant roles in vertebrate organogenesis and morphogenesis. FGFR3 is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia, one of the most common dwarfisms in humans, but the molecular mechanism remains elusive. In this study, we found that chondrocyte-specific deletion of BMP type I receptor a (Bmpr1a) rescued the bone overgrowth phenotype observed in Fgfr3 deficient mice by reducing chondrocyte differentiation. Consistently, using in vitro chondrogenic differentiation assay system, we demonstrated that FGFR3 inhibited BMPR1a-mediated chondrogenic differentiation. Furthermore, we showed that FGFR3 hyper-activation resulted in impaired BMP signaling in chondrocytes of mouse growth plates. We also found that FGFR3 inhibited BMP-2- or constitutively activated BMPR1-induced phosphorylation of Smads through a mechanism independent of its tyrosine kinase activity. We found that FGFR3 facilitate BMPR1a to degradation through Smurf1-mediated ubiquitination pathway. We demonstrated that down-regulation of BMP signaling by BMPR1 inhibitor dorsomorphin led to the retardation of chondrogenic differentiation, which mimicks the effect of FGF-2 on chondrocytes and BMP-2 treatment partially rescued the retarded growth of cultured bone rudiments from thanatophoric dysplasia type II mice. Our findings reveal that FGFR3 promotes the degradation of BMPR1a, which plays an important role in the pathogenesis of FGFR3-related skeletal dysplasia.

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Achondroplasia (ACH), the most common form of human dwarfism, is caused by an activating autosomal dominant mutation in the fibroblast growth factor receptor-3 (FGFR3) gene. Genetic overexpression of C-type natriuretic peptide (CNP), a positive regulator of endochondral bone growth, prevents dwarfism in mouse models of ACH. However, administration of exogenous CNP is compromised by its rapid clearance in vivo through receptor-mediated and proteolytic pathways. Using in vitro approaches, we developed modified variants of human CNP, resistant to proteolytic degradation by neutral endopeptidase (NEP), that retain the ability to stimulate signaling downstream of the CNP receptor, natriuretic peptide receptor B (NPR B). The variants tested in vivo demonstrated significantly longer serum half-lives than native CNP. Subcutaneous administration of one of these CNP variants, BMN 111, resulted in correction of the dwarfism phenotype in a mouse model of ACH and overgrowth of the axial and appendicular skeletons in wild-type mice without observable changes in trabecular and cortical bone architecture. Moreover, significant growth plate widening that translated into accelerated bone growth, at hemodynamically tolerable doses, was observed in juvenile cynomolgus monkeys that had received daily subcutaneous administrations of BMN 111. BMN 111 was well tolerated and represents a promising new approach for treatment of patients with ACH.
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as well as matrix production and degradation determines theheight of the growth plates. In humans, the cessation oflinear growth usually coincides with the end of pubertywhen growth plates become entirely replaced by bone.Similar to the appendicular skeleton, in the vertebrae,sternum and cranial base, bone growth occurs at synchon-droses—cartilaginous structures consisting of two opposedgrowth plates with a common zone of resting chondrocytes.As with endochondral growth plates, synchondroses alsobecome replaced by bone. The regulation of growth plateand synchondrosis closure is still not entirely understood.Endochondral ossification is controlled by multiple regulat-ory factors (1,2). An essential regulator of endochondral bonegrowth is fibroblast growth factor receptor 3 (Fgfr3). Fgfr3 ispreferentially expressed in proliferating and prehypertrophicchondrocytes in epiphyseal growth plates (3,4). Activatingmutations in FGFR3 cause autosomal dominant human skel-etal disorders, achondroplasia, thanatophoric dysplasia andhypochondroplasia (5–9). Thanatophoric dysplasia is themost common lethal skeletal dysplasia, and achondroplasiais the most common non-lethal form of dwarfism. Despiteits non-lethality, common and serious complications in achon-droplasia are a small foramen magnum and spinal stenosis(10,11) (Fig. 1). Stenosis of the foramen magnum, theorifice in the occipital bone through which passes the spinalcord from the medulla oblongata, has been associated withhydrocephalus and sudden death in infancy (12–14) as wellas headaches in older children (15). Currently, surgical enlar-gement of very small foramen magnum is recommended for,10% of children with achondroplasia (11,16). Narrowingof the spinal canal, which contains the spinal cord and caudaequina, is a common complication in adults with achondropla-sia and can cause neurologic deficits including myelopathy,radiculopathy and neurogenic claudication. In addition, insuf-ficient growth of the cranial base causes midface hypoplasia,which leads to obstructive sleep apnea, otitis media anddental malocclusion.Inadequate growth of the spinal canal, foramen magnumand cranial base in patients with FGFR3 mutations could bedue to deficient cell proliferation, hypertrophy and/or matrixproduction, and/or due to premature closure of synchondroses.Support for the latter mechanism comes from computedtomography (CT) studies in patients with achondroplasiawhere premature closure of occipital bone synchondroseswas observed (17). To explore the developmental mechanismsthat contribute to these complications, we examined synchon-droses of the spine and cranial base in human specimens fromchildren who died from homozygous achondroplasia andthanatophoric dysplasia, and we studied the timing of synch-ondrosis closure in mice with the Fgfr3 mutation G374R,which corresponds to the common human achondroplasiamutation, and in mice that express a constitutively activeform of MEK1, a downstream effector of Fgfr3 signaling.In humans and in mice, we observed premature closure ofmultiple synchondroses. Our results indicate that Fgfr3 andMAPK signaling in chondrocytes regulate synchondrosisclosure, osteoblast differentiation and bone formation, provid-ing novel insights into the developmental mechanisms ofspinal canal stenosis, foramen magnum stenosis and midfacehypoplasia in achondroplasia. If premature synchondrosisclosure accounts for spinal canal stenosis, foramen magnumstenosis andmidface hypoplasia,promoting treatment for these complications must startbefore the synchondroses close.then futuregrowth-RESULTSHuman specimens from homozygous achondroplasia andthanatophoric dysplasia were examined at the InternationalSkeletal Dysplasia Registry at Cedars-Sinai Medical Center.The synchondroses in the cranial base and lumbar vertebraewere examined in one case of homozygous achondroplasia,four cases of thanatophoric dysplasia (three perinatal, onefetal) and one control fetus (Table 1).Premature synchondrosis closure in homozygousachondroplasiaGross inspection and radiographic examination of the mid-sagittal slice of the cranial base in an infant who died fromhomozygous achondroplasia (Case 1) showed absence or pre-mature closure of the spheno-occipital synchondrosis (Fig. 2Band C), which normally closes between 11 and 25 years of age(18–22). We also examined the neurocentral synchondroses ofthe lumbar vertebrae that normally close between 3 and 14years of age (23–26). X-ray examination of the third lumbarspine showed complete fusion of the neurocentral synchondro-sis on one side and partial fusion on the other side (Fig. 2D),consistent with premature closure. Fusion was confirmedhistologically (data not shown).Premature synchondrosis closure in thanatophoricdysplasiaWe also examined one 27-week thanatophoric dysplasia fetuswith an R248C mutation (Case 2) and one 26-week gestationcontrol without any signs of skeletal dysplasia (Case 3). Radio-graphic examination showed narrowing of the foramenmagnum in the thanatophoric dysplasia fetus (Fig. 2E and F).Although the intraoccipital synchondroses were still openat this stage, this fetus had a bony bridge forming aroundthe anterior intraoccipital synchondroses (Fig. 2E) that wasFigure 1. Spinal canal stenosis in achondroplasia. Axial CT myelogram of thefifth lumbar spine shows the narrowing of the spinal canal in a 46-year-oldfemale achondroplasia patient with FGFR3 G380R mutation, who presentedwith paraparesis (right). The contrast medium injected into the subarachnoidspace was excluded in the achondroplasia patient due to severe spinal canalstenosis. (Left) 41-year-old female patient with irrelevant spinal disorder.White bars indicate 1 cm.228Human Molecular Genetics, 2009, Vol. 18, No. 2

sternum (Fig. 4C and data not shown). Fgfr3 was also weaklyexpressed in chondrocytes of the resting zone of the synchon-droses and some of the cells in the periosteum and bone spon-giosa. To examine the mechanism of premature synchondrosisclosure in Fgfr3G374R/þmice, we analyzed a series of vertebralsynchondroses harvested at various stages of development. Atembryonic day 16.5 (E16.5), no obvious difference wasobserved between wild-type embryos and Fgfr3G374R/þembryos (Fig. 4A). However, from postnatal day 1 (P1), theneurocentral synchondrosesFgfr3G374R/þmice. While the neurocentral synchondroses ofwild-type mice did not close until about P14 (data not shown),neurocentral synchondroses in Fgfr3G374R/þmice showedpartial closure at P4 and complete closure by P8. Interestingly,the growth plate-like structure of the neurocentral synchon-drosis consisting of the zones of the resting, proliferatingand hypertrophic chondrocytes was well maintained at P8 inwild-type mice (Fig. 4B). In contrast, Fgfr3G374R/þmice hadpremature loss of the zones of the resting and proliferatingchondrocytes beginning at P2. By P4, most of the chondrocytesbecame hypertrophic as evidenced by immunohistochemistryfor type X collagen (Fig. 4D).Because reduced cell proliferation, early apoptosis or preco-cious terminal differentiation could each be responsible for theprecocious synchondrosis closure, we examined proliferationof chondrocytes in the synchondrosis by bromodeoxyuridine(BrdU) incorporation, differentiation of chondrocytes byimmunohistochemistry for type X collagen and apoptosis byTUNEL assay. We also looked for evidence of vascular andosteoclast/chondroclast invasion by immunohistochemistryfor von Willebrand factor, MMP9, MMP13, tartrate-resistantacid phosphatase (TRAP) and in situ hybridization for Vegf.At E16.5, there was no difference in the number of BrdU-labeled cells in the neurocentral synchondrosis between wild-type and mutant mice (Fig. 4E). However, by P2, the mutantmice had fewer BrdU-labeled cells. In the wild-type synchon-drosis, numerous BrdU-labeled cells were found in the zonesthat correspond to the proliferative zone of growth plate,narrowedprogressively inwhile only a few chondrocytes were labeled with BrdU in thesynchondrosis of Fgfr3G374R/þmice. Immunohistochemistryfor an endothelial marker, von Willebrand factor, showedincreased vascular endothelial cells at the chondro-osseousjunction of synchondroses of Fgfr3G374R/þmice, suggestingincreased vascular invasion (Fig. 4F). In situ hybridizationanalysis showed increased Vegf expression in hypertrophicchondrocytes of Fgfr3G374R/þmice compared with wild-typemice, suggesting a role for VEGF in the increased vasculariza-tion (Fig. 4G). No obvious difference was found in the numberof osteoclasts/chondroclasts, apoptosis in synchondroses byTUNEL staining and expression of MMP9 and MMP13between Fgfr3G374R/þmice and wild-type littermate mice inthese analyses (data not shown).Increased bone formation in the perichondrium of micethat express Fgfr3 G374RSimilar to synchondroses in humans with thanatophoric dys-plasia and homozygous achondroplasia, Fgfr3G374R/þmicehad bone formation encasing the closing synchondroses. Toexamine osteoblast differentiation around the closing synchon-droses, we expressed the Col1a1-LacZ transgene in Fgfr3G374R/þmice. The Col1a1-LacZ transgene expresses LacZunder the control of a 2.3 kb osteoblast-specific promoter ofCol1a1, a gene for type I collagen (28). The neomycin cassettewas removed by using the Zp3-Cre transgene, so that the Fgfr3allele was recombined in a systemic fashion. X-gal staining ofFgfr3G374R/þ;Col1a1-LacZ mice showed a marked increase inosteoblasts around the synchondroses at P3 (Fig. 5A). We alsoexamined the expression of Runx2, a transcription factoressential for osteoblast differentiation (29,30). Runx2-positivecells were increased in the periosteum of mutant micecompared with wild-type mice at P4 (Fig. 5B). These obser-vations strongly suggest that increased Fgfr3 signalingpromotesosteoblastdifferentiationsynchondroses.aroundthe closingFigure 3. Skeletal preparations after alizarin red staining of mice that express Fgfr3 G374R and wild-type littermate mice at P10. Synchondroses in the cranialbase (A) and spine (B) were prematurely closed in mice that express Fgfr3 G374R. Arrows indicate fused synchondroses. Asterisks indicate the neurocentralsynchondroses. Scale bars indicate 1 mm. Abbreviations: Wt, wild-type mice, (G374R) mice that express Fgfr3 G374R; is, intersphenoid; so, spheno-occipital;aio, anterior intraoccipital; sc, spinal canal; C5, 5th cervical vertebrae; T8, eighth thoracic vertebrae; L4, fourth lumbar vertebrae.230 Human Molecular Genetics, 2009, Vol. 18, No. 2

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Figure 4. Premature loss of proliferating chondrocytes and increased vascular invasion in the synchondroses of mice that express Fgfr3 G374R. (A) Hematox-ylin, eosin and alcian blue-stained horizontal sections of the spine showed premature closure of the neurocentral synchondroses in mice that express Fgfr3G374R. Asterisk indicates neurocentral synchondroses and sc denotes spinal cord. (B) Higher magnification of the closing neurocentral synchondrosesbetween P2 and P8. All the vertebrae shown in (A) and (B) are either T12 or L1. (C) In situ hybridization of Fgfr3 of the spheno-occipital synchondrosis ofwild-type mice at P1. Fgfr3 is strongly expressed in chondrocytes in the proliferating and prehypertrophic zones. pi denotes pituitary glands. (D) Type X collagenimmunostaining of neurocentral synchondrosis of mice that express Fgfr3 G374R and wild-type littermate mice at P4. Mice that express Fgfr3 G374R showedpremature loss of the zones of resting and proliferating chondrocytes. (E) Immunostaining of the neurocentral synchondrosis for BrdU at E16.5 and P2. BrdU-labeled chondrocytes are markedly reduced in the closing neurocentral synchondrosis of mice that express Fgfr3 G374R at P2. (F) Immunostaining for vonWillebrand factor showed increased vascular endothelial cells at the chondro-osseous junction of the sternum of mice that express Fgfr3 G374R at P5. (G)In situ hybridization analysis showed increased Vegf expression in the synchondroses of the sternum of mice that express Fgfr3 G374R at P1.Human Molecular Genetics, 2009, Vol. 18, No. 2231